CN106086075B

CN106086075B - The construction method of CXCR4RNAi slow virus carrier

Info

Publication number: CN106086075B
Application number: CN201610462279.4A
Authority: CN
Inventors: 胡志坚; 陈愉生; 李鸿茹; 钟雪晶
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2016-06-23
Filing date: 2016-06-23
Publication date: 2019-08-06
Anticipated expiration: 2036-06-23
Also published as: CN106086075A

Abstract

The present invention provides a kind of construction method of CXCR4RNAi slow virus carrier, pass through the mRNA complete sequence according to GenBank CXCR4, the secondary structure of said target mrna sequence is assessed using 4.4 software of RNA structure after BLAST sequence analysis confirms specificity, obtains 19nt target sequence；Then it is single-stranded its corresponding two shRNA oligonucleotides to be synthesized for the design of each target sequence；Then after cyclic annular empty plasmid digestion, endonuclease bamhi connects PLVX.1 carrier and shRNA oligonucleotides double-strand, connect the recombinant vector formed, connection product converts fresh competent cell E.coli DH5 α, positive colony is chosen after 37 DEG C of culture 16h carries out bacterium colony PCR identification, after sequencing result verifies construction of recombinant plasmid success, recombinant plasmid is extracted.

Description

The construction method of CXCR4RNAi slow virus carrier

Technical field

The invention belongs to genetic engineering fields, and in particular to a kind of construction method of CXCR4RNAi slow virus carrier.

Background technique

Currently worldwide lung cancer morbidity rate rises year by year, and about dies of lung cancer more than a million people every year, As one of most common cause of the death of malignant tumour, there is cranium brain transfer, metastatic encephaloma Natural Survival in the course of disease in about 30% patient of lung cancer Time is 1~3 month, is one of malignant tumour severe complication, and cranium brain transfer is the one of the major reasons of lung cancer death.Chemotactic The factor is a kind of cell factor with biochemistry chemotaxis, and wherein SDF-1 and its specific receptor CXCR4 are thin in tumour It played an important role in the migration and invasion of born of the same parents.Researches show that the signal transduction that SDF-1 and its receptor CXCR 4 form is logical It road may be close with the generation, development and cranium brain transfer relationship of lung cancer.In non-small cell lung cancer early diagnosis and therapy, invasion It is the ineffective principal element of Treatment for Non-small Cell Lung with transfer.In order to preferably study CXCR4/SDF-1 biological axis Effect in non-small cell lung cancerous invasion and transfer especially brain metastes, this research are thin using the lung cancer with brain metastes potential Born of the same parents PC-9 constructs cell model, and the shRNA segment of CXCR4 gene order is directed to by designing, and constructs slow virus shRNA interference table Up to carrier, thus the expression of the targeted silent lung carcinoma cell CXCR4 gene of specificity.The spy of the shRNA interference of lentivirus mediated Point be it is efficient, stablize and silence durations it is long, for further research SDF-1/CXCR4 biological axis in non-small cell lung cancer cranium Effect in brain metastes provides tool.

Summary of the invention

The purpose of the present invention is to provide a kind of construction methods of CXCR4RNAi slow virus carrier.

To achieve the above object, the present invention adopts the following technical scheme:

CXCR4RNAi slow virus carrier, the carrier contain CXCR4 genetic fragment.

The construction method of CXCR4RNAi slow virus carrier includes the following:

(1) interference sequence designs: according to the mRNA complete sequence of GenBank CXCR4, confirming through BLAST sequence analysis special The secondary structure of said target mrna sequence is assessed using 4.4 software of RNA structure after the opposite sex, obtains 19nt target sequence； Then it is single-stranded its corresponding two shRNA oligonucleotides to be synthesized for the design of each target sequence；A pair of of control sequence is designed simultaneously siRNAc；

(2) building and identification of Lentiviral: empty carrier pGC-LV is marked with GFP, by PLVX-shRNA- PURO vector ring-type empty plasmid BamHAnd EcoRRestriction enzyme carries out double digestion, and recycles PLVX-SHRNA- PURO vector endonuclease bamhi, connection sky PLVX.1 carrier (coming from clontech company) and shRNA oligonucleotides double-strand, even The recombinant vector to be formed is connect, connection product converts fresh competent cell E.coli DH5 α, and positive gram is chosen after 37 DEG C of culture 16h Grand progress bacterium colony PCR identification extracts recombinant plasmid after sequencing result verifies construction of recombinant plasmid success；

(3) QPCR screens the primer that the QPCR primer of shRNA:CXCR4 gene is vector multiple cloning site both ends, culture Plasmid and lipo2000 compound are added in six orifice plates, 293 cell, extract the RNA of group of cells, RNA is inverted by 293 cells Record is cDNA, is template using cDNA, utilizes the relative expression quantity of the QPCR primer detection CXCR4 gene, fluorescent quantitation inspection Survey each group primer；PCR reaction condition: 95 DEG C of 30s, 1 circulation, 55 DEG C of 30s40 circulations, 95 DEG C of 5s, 60 DEG C of 1min, 95 DEG C of 15s.

QPCR primer sequence described in step (3) are as follows: Primer(+): 5 ' GGAGAGTTGTAGGATTCTAC-3 '^, Primer(-):5’-CCTCGGTGTAGTTATCTGAAG-3^’。

PLVX-shRNA-PURO vector ring-type empty plasmid is transformed by clontech company carrier PLVX-shRNA2 , concrete mode is: puro expression cassette is cloned into behind PLVX-shRNA2 carrier ZSgreen by amplification puro expression cassette.It is public The green hyperfluorescence albumen recognized, those skilled in the art can according to said method obtain the plasmid.

The present invention has the advantages that

Slow virus carrier and retroviral vector, adenovirus vector, gland relevant viral vector etc. are compared, and are had below Advantage: (1) can infect division cells and nondividing phase cell, such as nervous system, hemopoietic system nondividing phase cell, be immunized It reacts smaller；(2) transferable genetic fragment is larger, can be inserted into the genetic fragment of about 10 kb；(3) virus mutation and have multiple The viral probability of ability processed is smaller, greatly reduces virus (the replication competent of replication capacity Virus, RCV)；(4) target gene and host genome are by integrating the expression, it can be achieved that the target gene long period.Gene RNA perturbation technique in treating has become the new method of oncotherapy, and slow virus carrier can safely shift tumour target gene To body.By the RNAi technology of lentivirus-mediated, influence factor relevant to disease is found, and attempt thus to set about grinding The method for studying carefully treatment achievees the effect that treat disease.It should also ensure that higher safety, validity and reliability thus, and Clinical experimental stage gradually is entered from experiment in vitro and zoopery, it is believed that the technology will be in research field and clinic from now on There is broader prospect in terms of practical application.

Detailed description of the invention

Fig. 1 CXCR4-shRNA positive colony PCR qualification figure, 1: negative control (ddH₂O)；2:Marker；3-8: CXCR4-shRNA clone.

In 293 cell of Fig. 2 each group CXCR4 expression variation (, n=3), *P<0.01,**P=0.192>0.05)。

Specific embodiment

1 materials and methods

1.1 material

Human lung adenocarcinoma PC9 cell strain is purchased from Shanghai Yu Bo Biotechnology Co., Ltd.Cell DMEM 90%, Fetal 10% culture medium of Bovine Serum, at 37 DEG C, 5%CO₂Cell incubator in routine culture.CXCR4 rabbit polyclonal antibody, GAPDH mouse is mostly anti-to be purchased from abcam company, and the goat antirabbit secondary antibody of horseradish peroxidase-labeled is purchased from abcam company

1.2 method

1.2.1 interference sequence designs

According to the mRNA complete sequence (Accession No:NM_003467) of GenBank CXCR4, through BLAST homology ratio To applying 4.4 software of RNA structure to assess the secondary structure of said target mrna sequence after confirming specificity, 3 are obtained 19nt target sequence.Then it is single-stranded its corresponding two shRNA oligonucleotides to be synthesized for the design of each target sequence.A pair is designed simultaneously Control sequence siRNAc.Then it is single-stranded two shRNA oligonucleotides to be synthesized according to the sequence design of siRNA.It designs as follows: BamH+ 19 nt target nucleotide sequences of restriction enzyme site+loop-stem structure (TTCAAGAGA)+target sequence complementary series+RNA PolyPolymerization Enzyme transcription pausing site (TTTTTT)+EcoRSix regions of restriction enzyme site, be respectively designated as shRNA1, shRNA2, ShRNA3 and shRNAc, particular sequence are shown in Table 1.ShRNA oligonucleotide chain is synthesized by Shanghai Sangon Biotech Company.

3 specificity SiRNA target sequences of 1. CXCR4 gene of table

1.2.2 the building and identification of Lentiviral

Empty carrier pGC-LV is marked with GFP.By PLVX-shRNA-PURO vector ring-type empty plasmid BamH And EcoRRestriction enzyme carries out double digestion, and recycles PLVX-SHRNA-PURO vector endonuclease bamhi.Connection is empty PLVX.1 carrier and shRNA oligonucleotides double-strand, the recombinant vector for connecting formation are respectively labeled as: PLVX-CXCR4shRNA1, PLVX-CXCR4shRNA2,PLVX-CXCR4shRNA3,PLVX-CXCR4shRNAc.Connection product converts fresh competent cell E.coli DH5α.Positive colony is chosen after 37 DEG C of culture 16h and carries out bacterium colony PCR identification, is served the raw work in sea and is carried out DNA sequencing identification. After sequencing result verifies construction of recombinant plasmid success, recombinant plasmid is extracted.

1.2.3 QPCR screens the shRNA of optimum jamming effect

The QPCR primer of CXCR4 gene is the primer at vector multiple cloning site both ends, sequence Primer(+): 5, ' GGAGAGTTGTAGGATTCTAC3^’, Primer (-): 5^’CCTCGGTGTAGTTATCTGAAG3^’,

293 cells are cultivated, by plasmid (pLVX-CXCR4shRNA1, pLVX-CXCR4shRNA2, pLVX- CXCR4shRNA3, pLVX-acGFP-C1) and lipo2000 compound be added six orifice plates, 293 cell in, extract group of cells RNA reverse transcription is cDNA by RNA, is template using cDNA, is detected the relative expression quantity of CXCR4 gene, fluorogenic quantitative detection is each Group primer.PCR reaction condition: 95 DEG C of 30s, 1 circulation, 55 DEG C of 30s40 circulations, 95 DEG C of 5s, 60 DEG C of 1min, 95 DEG C of 15s.With 2^-△△CtValue indicates the relative expression levels of CXCR4 gene mRNA.

1.2.4 slow virus is packed

According to interference screening as a result, by the interference carrier PLVX-CXCR4-shRNA1 coated plate of optimum jamming effect, 37 DEG C Overnight incubation, picking single colonie are inoculated into the LB culture medium of 200ml, 250rpm, and 37 DEG C are shaken bacterium overnight incubation, a large amount of to extract Plasmid.Mass propgation 293T cell, the day before transfection are anti-with being free of with the 293T cell of trypsin digestion logarithmic growth phase The DMEM complete medium inoculation 293FT cell of raw element is in 60 mm plates, 2 × 106 cells of each ware inoculating cell, and 37 DEG C, 24 h are cultivated in 5% CO2 incubator.By shRNAc, CXCR4-shRNA respectively with slow virus packaging plasmid cotransfection 293T Cell.48 h collect the 293T cell supernatant after transfection, and filtrate is concentrated after ultracentrifugation, remove supernatant, and it is following heavy to retain It forms sediment, suitable PBS lytic virus precipitating is added, is stored in after packing in viral pipe, -80 DEG C of long-term preservations.

1.2.5 virus titer measures

293 cells are cultivated, seed cells into 48 orifice plates, 105, every hole cell, 37 DEG C, 5%CO2 continues to cultivate 18h.It takes The viral dilution of 20 μ l is to the DMEM culture medium of 200 μ l, and successively 10 dilution viruses are (10^-3、10^-4、10^-5、10^-6、10^-7), it mixes Even diluted virus, is added separately in 48 holes, and every 100 μ l of hole, 37 DEG C, 5%CO2 continues to cultivate 48h.It is inverted using fluorescence micro- The quantity of each pore area fluorecyte of mirror detection statistics, calculate virus titer in conjunction with extension rate: titre (TU/m1)=fluorescence is thin The effective extension rate of born of the same parents' number X.

1.2.6 the foundation of stable interference cell strain

It infects preceding 1 day logarithmic growth phase PC9 cell in good condition and presses every 5 x10 of hole⁵It is a to be inoculated in 6 orifice plates.Infection When, slow virus is diluted according to MOI=100, is separately added into hole containing 8 mgL^-12 kinds of dilution restrovirus liquid 1mL of polybrene, The target cell of blank is concurrently set as control.After cultivating 16h, it is changed to fresh DMEM complete medium 2mL and continues to cultivate, Fluorescence microscopy microscopic observation efficiency of infection after 48h.Single cell suspension is made after metainfective cell is digested with pancreatin, according to every The density that ware is 1000 is inoculated in the culture dish of 10cm, and culture selects monoclonal cell again to monoclonal cell group is formed It is cloned, then picking sufficient amount monoclonal cell carries out amplification cultivation, prepares for detection in next step.Obtained stabilization Interference cell is respectively designated as PC9/CXCR4-shRNA, and negative control cell is named as PC9/shRNAc.

1.2.7 QPCR detects cell PC-9mRNA jamming effectiveness

PC9/CXCR4-shRNA, PC9/shRNAc cell are collected, by QIAGEN Rneasy Mini kit kit explanation Experimental method and step in book extract cell total rna, and according to reverse transcription reagent box (PrimeScript RT reagent Kit the synthesis of cDNA reverse transcription) is carried out.Using this cDNA as template, PCR amplification, CXCR4 are carried out using PCR kit for fluorescence quantitative It is synthesized with GAPDH gene (internal reference) primer by Sangon Biotech's design.CXCR4:Primer (+): 5^, GGAGAGTTGTAGGATTCTAC3,,Primer(-):5^,CCTCGGTGTAGTTATCTGAAG3^,.PCR reacts item Part: 95 DEG C of 30s, 1 circulation, 55 DEG C of 30s40 circulations, 95 DEG C of 5s, 60 DEG C of 1min, 95 DEG C of 15s.CXCR4 base is indicated with 2^-ddCt value Because of the relative expression levels of mRNA.

1.2.8 Western blot detects cell PC-9 albumen jamming effectiveness

PC9/CXCR4-shRNA, PC9/shRNAc cell are collected, cell pyrolysis liquid is added and cracks 10 min on ice, takes egg Bai Shangqing BCA determination of protein concentration concentration.50~80g protein is taken to carry out 10%SDS-PAGE electrophoresis, the egg after separation On white matter electrotransfer to pvdf membrane, 2 h are closed with the confining liquid room temperature containing 5% skimmed milk power；The diluted rabbit-anti of 1:2000 is added People CXCR4 polyclonal antibody and the diluted mouse anti human GAPDH monoclonal antibody of 1:2000,4 DEG C of reactions are overnight；TBS washes film Afterwards, it is separately added into the diluted secondary antibody of 1:3000 (goat antirabbit or goat anti-mouse IgG of HRP label), reacts at room temperature 2h； TBS After washing, chemical illuminating reagent, cold CCD imaging system exposure, development and fixing is added.Scan film, to each protein band into The analysis of row gray value indicates CXCR4 with the ratio of CXCR4 protein band gray value and internal reference GAPDH protein band gray value The relative expression levels of albumen.

2 results

2.1 the PCR of recombinant slow virus plasmid and sequencing identification

PCR identify recombinant slow virus plasmid when, connect into shRNA segment positive colony PCR fragment size be 200bp. PCR product is subjected to electrophoresis, the 1st, 2,3 pair of target sequence result shows positive colony (Fig1).From each target sequence It respectively selects one in positive colony to be sequenced, as a result test result display sequence is correct.

The shRNA of 2.2 optimum jamming effects is screened

The mRNA expression of CXCR4 in 293 cell of each group is as shown in Fig. 2, as the result is shown: passing through single factor test variance point Analysis, compared with blank control group, the expression of the CXCR4 of 293 cells in experimental group be remarkably decreased (P=0.0001 < 0.01), the CXCR4 expression of 293 cells of negative control vector group changes not statistically significant (P=0.192 > 0.05), says Bright CXCR4-shRNA carrier can significantly lower the expression of CXCR4mRNA in 293 cells, and CXCR4-shRNA1 carrier is lowered most Obviously.

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>Medical University Of Fujian

<120>construction method of CXCR4RNAi slow virus carrier

<130> 10

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213>artificial sequence

<400> 1

ggagagttgt aggattctac 20

<210> 2

<211> 21

<212> DNA

<213>artificial sequence

<400> 2

cctcggtgta gttatctgaa g 21

<210> 3

<211> 59

<212> DNA

<213>shRNA1(Top)

<400> 3

gatccggatc agcatcgact ccttttcaag agaaaggagt cgatgctgat ccttttttg 59

<210> 4

<211> 59

<212> DNA

<213>shRNA1(Bottom)

<400> 4

aattcaaaaa aggatcagca tcgactcctt tctcttgaaa aggagtcgat gctgatccg 59

<210> 5

<211> 59

<212> DNA

<213>shRNA2(Top)

<400> 5

gatccggatc agtatataca cttcttcaag agagaagtgt atatactgat ccttttttg 59

<210> 6

<211> 59

<212> DNA

<213>shRNA2(Bottom)

<400> 6

aattcaaaaa aggatcagta tatacacttc tctcttgaag aagtgtatat actgatccg 59

<210> 7

<211> 59

<212> DNA

<213>shRNA3(Top)

<400> 7

gatccgcaag gcagtccatg tcatttcaag agaatgacat ggactgcctt gcttttttg 59

<210> 8

<211> 59

<212> DNA

<213>shRNA3(Bottom)

<400> 8

aattcaaaaa agcaaggcag tccatgtcat tctcttgaaa tgacatggac tgccttgcg 59

<210> 9

<211> 59

<212> DNA

<213>shRNAc(Top)

<400> 9

ccggtgcttc gacatttaac caatttcaag agaattggtt aaatgtcgaa gcttttttg 59

<210> 10

<211> 59

<212> DNA

<213>shRNAc(Bottom)

<400> 10

aattcaaaaa agcttcgaca tttaaccaat tctcttgaaa ttggttaaat gtcgaagca 59

Claims

The construction method of 1.CXCR4RNAi slow virus carrier, it is characterised in that: the construction method includes the following:

(1) interference sequence designs: according to the mRNA complete sequence of GenBank CXCR4, confirming specificity through BLAST sequence analysis The secondary structure of said target mrna sequence is assessed using 4.4 software of RNA structure afterwards, obtains 19nt target sequence；Then It is single-stranded that its corresponding two shRNA oligonucleotides is synthesized for the design of each target sequence；

It is as follows that each target sequence design synthesizes its corresponding two shRNA oligonucleotides single stranded sequence:

ShRNA1-Top:gatccGGATCAGCATCGACTCCTTTTCAAGAGAAAGGAGTCGATG CTGATCCTTTTTTg,

ShRNA1-Bottom:aattcAAAAAAGGATCAGCATCGACTCCTTTCTCTTGAAAAG GAGTCGATGCTGATC Cg；And

ShRNA2- Top:gatccGGATCAGTATATACACTTCTTCAAGAGAGAAGTGTATATACTGATCC TTTTTTg,

ShRNA2- Bottom:aattcAAAAAAGGATCAGTATATACACTTCTCTCTTGAAGAAGTGTATA TACTGATC Cg；And

ShRNA3-Top:gatccGCAAGGCAGTCCATGTCATTTCAAGAGAATGACATGGACT GCCTTGCTTTTTTg,

ShRNA3-Bottom:aattcAAAAAAGCAAGGCAGTCCATGTCATTCTCTTGAAATG ACATGGACTGCCTTG Cg；

(2) building and identification of Lentiviral: empty carrier pGC-LV is marked with GFP, by PLVX-shRNA-PURO Vector ring-type empty plasmid, uses BamHAnd EcoRRestriction enzyme carries out double digestion, and recycles PLVX-SHRNA-PURO Vector endonuclease bamhi, connection sky PLVX.1 carrier and shRNA oligonucleotides double-strand, connect the recombinant vector of formation, and connection produces Object converts fresh competent cell E.coli DH5 α, and positive colony is chosen after 37 DEG C of culture 16h and carries out bacterium colony PCR identification, sequencing knot After fruit verifies construction of recombinant plasmid success, recombinant plasmid pLVX-CXCR4shRNA is extracted；

(3) QPCR screens the primer that the QPCR primer of shRNA:CXCR4 gene is vector multiple cloning site both ends, and culture 293 is thin Plasmid pLVX-CXCR4shRNA and the lipo2000 compound of acquisition is added in six orifice plates, 293 cell born of the same parents, and it is thin to extract each group RNA reverse transcription is cDNA, is template using cDNA, utilizes the opposite of the QPCR primer detection CXCR4 gene by the RNA of born of the same parents Expression quantity, fluorogenic quantitative detection each group primer；PCR reaction condition: 95 DEG C of 30s, 1 circulation, 55 DEG C of 30s40 circulations, 95 DEG C of 5s, 60 DEG C 1min, 95 DEG C of 15s.
2. the construction method of CXCR4RNAi slow virus carrier according to claim 1, it is characterised in that: institute in step (3) State QPCR primer sequence are as follows: Primer(+): 5 ' GGAGAGTTGTAGGATTCTAC-3 '^,Primer(-):5’- CCTCGGTGTAGTTATCTGAAG-3^’。
3. the construction method of CXCR4RNAi slow virus carrier according to claim 1, it is characterised in that: the PLVX- ShRNA-PURO vector ring-type empty plasmid is that carrier PLVX-shRNA2 transformation obtains.
4. the carrier that construction method building as described in claim 1 obtains.